Recombinant double-stranded RNA phage, and use of the same

ABSTRACT

A recombinant double stranded RNA (dsRNA) phage expresses dsRNA-encoded genes in eukaryote cells. Recombinant dsRNA phage are useful for the expression of dsRNA expression cassettes encoding passenger genes, such as, but not restricted to, vaccine antigens, bioactive proteins, immunoregulatory proteins, antisense RNAs, and catalytic RNAs in eukaryotic cells or tissues. Methods are provided to deliver recombinant dsRNA phage to eukaryotic cells and tissues, either by direct administration, formulated in lipid or polylactide-coglycolide, or by utilizing a bacterial vaccine vector.

This application claims priority to U.S. Provisional Patent ApplicationNo. 60/404,806 filed on Aug. 20, 2002. The complete contents of thatapplication are herein incorporated by reference.

This invention was made with the support of a grant from the NationalInstitutes of Health (NIH) grant numbers R01-A14194 and R01-055367. TheU.S. government has certain rights in this invention.

FIELD OF INVENTION

The present invention provides recombinant double stranded RNA (dsRNA)phage that express dsRNA-encoded genes in eukaryote cells. RecombinantdsRNA phage are useful for the expression of dsRNA expression cassettesencoding passenger genes, such as, but not restricted to, vaccineantigens, bioactive proteins, immunoregulatory proteins, antisense RNAs,and catalytic RNAs in eukaryotic cells or tissues. Methods are providedto deliver recombinant dsRNA phage to eukaryotic cells and tissues,either by direct administration, formulated in lipid orpolylactide-coglycolide, or by utilizing a bacterial vaccine vector.

BACKGROUND Double Stranded Ribonucleic Acid Phage

Double stranded RNA phage (herein “dsRP”) are atypical compared to otherRNA and DNA phage, and more closely resemble members of the reoviridaefamily [1–5]. The distinguishing attributes of dsRP are a genomecomprised of three double-stranded RNA (herein “dsRNA”) segments [2–4,6]and a lipid-containing membrane coat [7–12].

The genomic segments are contained within the nucleocapsid core, whichis comprised of the proteins P1, P2, P4, and P7, and is produced bygenes encoded on the 7051 bp dsRNA segment, designated “segment L”(GeneBank Accession # AF226851). Synthesis of positive-strand RNA(herein “mRNA”) occurs within the nucleocapsid, which is carried out byRNA-dependent RNA polymerase that may be encoded by gene 2 on segment L,based on sequence similarity to other bacterial RNA polymerases [4,13].However, gene 7 on segment L also plays a pivotal role in mRNA synthesis[5].

DsRP phi-6, the archetype of this family of dsRNA phage, normallyinfects Pseudomonas syringae [5], however, more recently isolated dsRPphi-8, phi-11, phi-12 and phi-13 can replicate to some extent inEscherichia coli strain JM109 (American type tissue culture collection(herein “ATCC”) # 53323) and O-antigen negative mutants of Salmonellaenterica serovar Typhimurium (herein designated “S. typhimurium”)[5,14–16].

By inserting a kanamycin-resistance allele into the M-segment of a dsRP,carrier strains were established and maintained [17]. Through thisapproach, several of the dsRPs were found to be capable of establishinga carrier state in host cells, in which infectious phage arecontinuously produced by the carrier strain [17]. The plaque-formingcapacity of the phage produced by the carrier strains is maintained forthree-five plate passages; however, after additional passages thenascent phage no longer formed plaques on the carrier strain, yetlow-levels of infectious phage were still produced [17]. In someinstances, a significant number of carrier strains lost the ability toproduce infectious phage all together, yet phage dsRNA segments werecontinuously maintained in the cytosol of such carrier bacteria. ThedsRNA from such bacterial strains displayed deletions in one of more ofthe segments [17]. In one instance a mutant phage lacking the segment-Swas isolated from one such carrier strain that had lost the capacity toproduce phage [17,18].

The life cycle of the dsRP phi-6 in bacteria has been described [5,11].Archetype dsRP phi-6 infects host cells by binding to the pilus. Thephage then uses the pilus to allow contact with the host cell membrane,thereby resulting in fusion and introduction of the nucleocapsid intothe periplasm. The nucleocapsid then is transported into the cytoplasm,an event that requires the endopeptidase activity of protein P5 and thetransporting property of protein P8. Interestingly, nucleocapsids thatbear a complete P8 shell are capable of spontaneous entry into bacterialprotoplasts, resulting in auto-transfection of the bacterial strain fromwhich the protoplasts were prepared [19,20].

Upon entering the cytoplasm, P8 is shed and the remaining nucleocapsid,which contains the three dsRNA segments and possesses RNA-dependent RNApolymerase activity, begins to synthesize mRNA copies of the dsRNAsegments L, M and S (FIG. 1). The proteins produced by segment L aremainly associated with procapsid production; segment M is mainlydedicated to the synthesis of the attachment proteins and the segment Sproduces the procapsid shell protein (P8), the lytic endopeptidase (P5),and the proteins (P9 and P12) involved in the generation of the lipidenvelope [12] (FIG. 1). Packaging of the dsRNA segments occurs insequential manner, whereby segment S is recognized and taken up by emptyprocapsids; procapsids containing segment S no longer binds this segmentbut now are capable of binding and taking up segment M; procapsids thatcontain segments S and M no longer bind these segments but now arecapable of binding and taking up segment L, resulting in the generationof the nucleocapsid. Once the nucleocapsid contains all threesingle-stranded RNA (herein “ssRNA”) segments synthesis of the negativeRNA strands begins to produce the dsRNA segments. The nucleocapsid thenassociates with proteins 5 and 8 (FIG. 1) and finally is encapsulated inthe lipid membrane, resulting the completion of phage assembly. Lysis ofthe host cell is thought to occur through the accumulation of themembrane disrupter protein P10, a product of segment M and requires theendopeptidase P5 [5].

The assembly of and RNA polymerase activity in dsRP procapsids does notrequire host proteins, as procapsids purified from an E. coli JM109derivative that expressed a cDNA copy of segment L are capable ofpackaging purified ssRNA segments L, M and S [5,19–24]. Following uptakeof the ssRNA segments in the above in vitro system, addition ofribonucleotides resulted in negative strand synthesis and the generationof the mature dsRNA segments [5,19–24]. Furthermore, after thecompletion of dsRNA synthesis P8 associates with nucleocapsids and asindicated above the resultant product is capable of entering bacterialprotoplasts and producing a productive infection [19,20].

Introduction of Nucleic Acids into Eukaryotic Cells

There are several techniques for introducing nucleic acids intoeukaryotic cells cultured in vitro. These include chemical methods(Felgner et al, Proc. Natl. Acad. Sci., USA, 84:7413–7417 (1987);Bothwell et al, Methods for Cloning and Analysis of Eukaryotic Genes,Eds., Jones and Bartlett Publishers Inc., Boston, Mass. (1990), Ausubelet al, Short Protocols in Molecular Biology, John Wiley and Sons, NewYork, N.Y. (1992); and Farhood, Annal. N.Y. Acad. Sci., 716:23–34(1994)), use of protoplasts (Bothwell, supra) or electrical pulses(Vatteroni et al, Mutn. Res., 291:163–169 (1993); Sabelnikov, Prog.Biophys. Mol. Biol, 62: 119–152 (1994); Brothwell et al, supra; andAusubel et al, supra), use of attenuated viruses [25–34](Moss, Dev.Biol. Stan., 82:55–63 (1994); and Brothwell et al, supra), as well asphysical methods (Fynan et al, supra; Johnston et al, Meth. Cell Biol,43(Pt A):353–365 (1994); Brothwell et al, supra; and Ausubel et al,supra).

Successful delivery of nucleic acids to animal tissue has been achievedby cationic liposomes (Watanabe et al, Mol. Reprod. Dev., 38:268–274(1994)), direct injection of naked DNA or RNA into animal muscle tissue(Robinson et al, Vacc., 11:957–960 (1993); Hoffman et al, Vacc.12:1529–1533; (1994); Xiang et al, Virol., 199:132–140 (1994); Websteret al, Vacc., 12:1495–1498 (1994); Davis et al, Vacc., 12:1503–1509(1994); and Davis et al, Hum. Molec. Gen., 2:1847–1851 (1993); [35,36]),and embryos (Naito et al, Mol. Reprod. Dev., 39:153–161 (1994); andBurdon et al, Mol. Reprod. Dev., 33:436–442 (1992)), intramuscularinjection of self replicating RNA vaccines [25–28,35,36] or intradermalinjection of DNA using “gene gun” technology (Johnston et al, supra).

Translation of mRNA into Protein in Eukaryotes and Prokaryotes

The ribosomal binding site (herein “RBS”) is the site recognized by theribosome for binding to the 5-prime (herein designated “5′”) end ofmRNA) molecules. This binding is essential for the translation of mRNAinto a protein by the ribosome. In prokaryotes, a defined RBS in the 5′end of the mRNA molecule that bears a sequence that is complementary tothe 3′ end of the small ribosomal RNA molecule (5S rRNA) (Chatteji etal, Ind. J. Biochem. Biophys., 29:128–134 (1992); and Darnell et al,supra; Lewin, supra; Watson et al, supra; and Watson et al, supra).Thus, in prokaryotes the RBS promotes association of the ribosome withthe 5′ end of the nascent mRNA molecule, whereupon translation isinitiated at the first initiation codon encountered (i.e. normally themethionine codon AUG) by the mRNA-associated ribosome (Darnell et al,supra; Lewin, supra; Watson et al, supra; and Alberts et al, supra). Atpresent, no such recognition pattern has been observed in the 5′eukaryotic mRNA-ribosome interactions (Eick et al, supra). In addition,prior to initiation of translation of eukaryotic mRNA, the 5′ end of themRNA molecule is “capped” by addition of methylated guanylate to thefirst mRNA nucleotide residue (Darnell et al, supra; Lewin, supra;Watson et al, supra; and Alberts et al, supra). It has been proposedthat recognition of the translational start site in mRNA by theeukaryotic ribosomes involves recognition of the cap, followed bybinding to specific sequences surrounding the initiation codon on themRNA. It is possible for cap independent translation initiation to occurand/or to place multiple eukaryotic coding sequences within a eukaryoticexpression cassette if a internal ribosome entry site (herein “IRES”)sequence, such as the cap-independent translation enhancer (hereindesignated “CITE”) derived from encephalomyocarditis virus (Duke et al,J. Virol., 66:1602–1609 (1992)), is included prior to, or between, thecoding regions. However, the initiating AUG codon is not necessarily thefirst AUG codon encountered by the ribosome (Louis et al, Molec. Biol.Rep., 13:103–115 (1988); and Voorma et al, Molec. Biol. Rep., 19:139–145(1994); Lewin, supra; Watson et al, supra; and Alberts et al, supra).Thus, RBS sequences in eukaryotes are sufficiently divergent from thatof prokaryotic RBS such that the two are not interchangeable.

Delivery of Nucleic Acids to Eukaryotic Cells

The commercial application of nucleic acid delivery technology toeukaryotic cells is broad and includes delivery of vaccine antigens(Fynan et al, Proc. Natl. Acad. Sci., USA, 90:11478–11482 (1993)),immunotherapeutic agents, and bioactive proteins designed to remedygenetic disorders (Darris et al, Cancer, 74(3 Suppl.):1021–1025 (1994);Magrath, Ann. Oncol., 5(Suppl 1):67–70 (1994); Milligan et al, Ann. NYAcad. Sci., 716:228–241 (1994); Schreier, Pharma. Acta Helv., 68:145–159(1994); Cech, Biochem. Soc. Trans., 21:229–234 (1993); Cech, Gene,135:33–36 (1993); Long et al, FASEB J., 7:25–30 (1993); and Rosi et al,Pharm. Therap., 50:245–254 1991)).

The delivery of nucleic acids to animal tissue for gene therapy hasshown significant promise in experimental animals and volunteers,particularly where a transient effect is required (Nabel, Circulation,91:541–548 (1995); Coovert et al, Curr. Opin. Neuro., 7:463–470 (1994);Foa, Bill. Clin. Haemat., 7:421–434 (1994); Bowers et al, J. Am. Diet.Assoc., 95:53–59 (1995); Perales et al, Eur. J. Biochem., 226:255–266(1994); Danko et al, Vacc., 12:1499–1502 (1994); Conry et al, Canc.Res., 54:1164–1168 (1994); and Smith, J. Hemat., 1:155–166 (1992)).Recently, naked DNA vaccines carrying eukaryotic expression cassetteshave been used to successfully immunize against influenza both inchickens (Robinson et al, supra) and ferrets (Webster et al, Vacc.,12:1495–1498 (1994)); against Plasmodium yoelii in mice (Hoffman et al,supra); against rabies in mice (Xiang et al, supra); against humancarcinoembryonic antigen in nice (Conry et al, supra) and againsthepatitis B in mice (Davis et al, supra). These observations open theadditional possibility that delivery of nucleic acids to eukayotictissue could be used for both prophylactic and therapeutic applications,wherein the prophylactic application has a significant impact in themortality and/or morbidity of the infectious agent, autoimmune diseaseor tumor prior to the acquisition of overt clinical disease, and thetherapeutic application has a significant impact in the mortality and/ormorbidity of the infectious agent, autoimmune disease or tumor followingthe development of overt clinical disease.

Therefore, there is a need to deliver eukaryotic expression cassettes,encoding endogenous or foreign genes that are vaccines or therapeuticagents to eukaryotic cells or tissue.

SUMMARY OF THE INVENTION

The present invention describes a novel and unexpected finding that dsRPare capable of delivering dsRNA eukaryotic expression cassettes toeukaryotic cells and tissue.

Heretofore, there has been no documented demonstration of dsRP invadingeukaryotic cells and introducing a eukaryotic expression cassette(s),which then is translated by the infected cells and progeny thereof. Thatis, the present invention provides the first documentation of functionalgenetic exchange between dsRP and eukaryotic cells.

This invention provides recombinant dsRP that express dsRNA-encodedgenes in eukaryote cells encoding a functional eukaryotic translationexpression cassettes. The prior art teaches the biology of dsRP inprokaryotic cells, such as P. syringae, E. coli, and S. typhimurium. ThemRNAs produced by dsRP are poorly translated in eukaryotic cells.Surprisingly, we found that the incorporation of cap-independenteukaryotic translation, herein referred to as “CITE” (also known as aninternal ribosome entry site, herein referred to as “IRES”) sequencesinto dsRP enables expression in eukaryotic cells or tissues. CITEsequences are discussed in detail in U.S. Pat. No. 6,500,419 to Hone,and the complete contents thereof is herein incorporated by reference.As will be shown in more detail below the IRES sequence and a passengergene of interest can be inserted into one or more of the three dsRNAsegments in the dsRP [17]. The resultant recombinant dsRP carrying arecombinant segment or segments produces messenger RNA in eukaryoticcells that is recognized by the eukaryotic translation apparatus (Seeexample below). The ensuing translation by the eukaryotic cell ribosomesresults in the expression of the passenger gene of interest.

Another object of this invention describes recombinant dsRP that carryalpha virus expression cassettes, such as but not restricted to thesemliki forest virus [29–34] or venezuelan equine encephalitis (hereindesignated “VEE”) virus [25–28], that are capable of self-amplification.

In yet another object of the current invention, methods are provided forthe administration of recombinant dsRP to eukaryotic cells and tissues,and the use of recombinant dsRP to induce an immune response or to causea biological affect in a target cell population.

In a still further object of the current invention, compositions andmethods are described for the delivery of dsRP to mammalian cells andtissues using bacterial vectors, and the use of said bacterial vectorscarrying recombinant dsRP to induce an immune response or to cause abiological affect in a target cell population.

In another embodiment, the present invention relates to live bacteriathat carry a recombinant dsRP containing one or more eukaryotictranslation expression cassettes encoding dsRNA encoding IRES sequencesthat are functionally linked to one or more passenger genes.

In yet another embodiment of this invention, recombinant dsRPcompositions are provided that incorporate an alphavirus expressioncassette into said dsRP, thereby harnessing the mRNA-amplifyingproperties of said alpha virus, resulting in the generation of dsRP thatare capable of substantively amplifying the mRNA of a passengerRNA-encoded gene in eukaryotic cells.

These and other objects of the present invention, which will be apparentfrom the detailed description of the invention provided hereinafter,have been met in one embodiment by providing compositions and methodsfor introducing and expressing a gene into eukaryotic cells, comprisinginfecting said cells with a recombinant dsRP carrying a eukaryotictranslation expression cassette comprised of dsRNA sequences encoding anIRES and the green fluorescent protein (herein designated “GFP”),wherein said dsRP carrying said eukaryotic translation expressioncassette is capable of expressing GFP in eukaryotic cells.

In another embodiment, the present invention relates to live bacteriathat carry a recombinant dsRP containing one or more eukaryotictranslation expression cassettes encoding dsRNA encoding IRES sequencesthat are functionally linked to one or more passenger genes.

In yet another embodiment of this invention, recombinant dsRPcompositions are provided that incorporate an alphavirus expressioncassette into said dsRP, thereby harnessing the mRNA-amplifyingproperties of said alpha virus, resulting in the generation of dsRP thatare capable of substantively amplifying the mRNA of a passengerRNA-encoded gene in eukaryotic cells.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic drawing showing the replication of dsRPnucleocapsids in bacterial cytoplasm.

FIG. 2 is a schematic drawing illustrating cloning cDNA copies of themRNA produced by dsRP.

FIG. 3 is a schematic drawing illustrating the construction ofrecombinant dsRP segments using cDNA clones.

FIG. 4 is a schematic drawing illustrating the generation of recombinantdsRP nucleocapsids.

FIG. 5 is a schematic representation of rdsRP-1 segment-S.

FIG. 6 is a schematic representation of the recombinant segment-S in aself-amplifying rdsRP.

DETAILED DESCRIPTION OF THE INVENTION

As mentioned above in one embodiment of the present inventionrecombinant dsRP (herein referred to as “rdsRP”) are provided thatexpress dsRNA-encoded genes in eukaryote cells. Normally, dsRP-encodedgenes are poorly translated in eukaryotic cells due to the lack ofcap-independent eukaryotic translation signaling sequences that arenecessary to launch efficient ribosome binding and the translation ofmRNA sequences into protein. Below rdsRP are provided that produce mRNAmolecules containing the appropriate translation initiation sequencesthat enable efficient recognition and translation in eukaryotic cells.It is surprising that only a simple modification to a prokaryotic virus(i.e. dsRP) results in efficient expression in a eukaryotic cell. Thisfinding suggests that a partial evolutionary leap by a virus fromprokaryote to eukaryote only requires the acquisition of small amountsof genetic information.

Recombinant DNA Techniques

The recombinant DNA procedures used in the construction of the followingrdsRP, including PCR, restriction endonuclease (herein referred to as“RE”) digestions, DNA ligation, agarose gel electrophoresis, DNApurification, and dideoxynucleotide sequencing, are described elsewhere[37–40](Miller, A Short Course in Bacterial Genetics, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1992); Bothwell et al,supra; and Ausubel et al, supra), bacteriophage-mediated transduction(de Boer, supra; Miller, supra; and Ausubel et al, supra), or chemical(Bothwell et al, supra; Ausubel et al, supra; Felgner et al, supra; andFarhood, supra), electroporation (Bothwel et al, supra; Ausubel et al,supra; and Sambrook, Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y.) and physicaltransformation techniques (Johnston et al, supra; and Bothwell, supra).The genes can be incorporated on phage (de Boer et al, Cell, 56:641–649(1989)), plasmids vectors (Curtiss et al, supra) or spliced into thechromosome (Hone et al, supra) of the target strain.

Gene sequences can be made synthetically using an Applied BiosystemsABI™ 3900 High-Throughput DNA Synthesizer (Foster City, Calif. 94404U.S.A.) and procedures provided by the manufacturer. To synthesize largesequences i.e greater than 200 bp, a series of segments of thefull-length sequence are generated by PCR and ligated together to formthe full-length sequence using procedures well know in the art [41–43].However, smaller sequences, i.e. those smaller than 200 bp, can be madesynthetically in a single round using an Applied Biosystems ABI™ 3900High-Throughput DNA Synthesizer (Foster City, Calif. 94404 U.S.A.) andprocedures provided by the manufacturer.

Recombinant plasmids are introduced into bacterial strains byelectroporation using a BioRad Gene-Pulser® set at 200Ω, 25 μF and 2.5kV (BioRad Laboratories, Hercules, Calif.) [38]. Nucleotide sequencingto verify cDNA sequences is accomplished by standard automatedsequencing techniques (Applied Biosystems automated sequencer, model373A). DNA primers for DNA sequencing and polymerase chain reaction(herein referred to as “PCR”) are synthesized using an AppliedBiosystems ABI™ 3900 High-Throughput DNA Synthesizer (Foster City,Calif. 94404 U.S.A.).

Source of IRES Sequences

mRNA molecules lacking a 5′ cap modifier, which is normally added in thenucleus to nuclear mRNA transcripts and enhances ribosome recognition,are poorly translated in eukaryotic cells unless an IRES sequence ispresent upstream of the gene of interest. The particular IRES employedin the present invention is not critical and can be selected from any ofthe commercially available vectors that contain IRES sequences. Thus,IRES sequences are widely available and can be obtained commerciallyfrom plasmid pIRES2-EGFP (Clontech; [44]) by PCR using primers specificfor the 5′ and 3′ ends of the IRES located at nucleotides 665–1251 inpIRES2-EGFP. The sequences in plasmid pIRES-EGFP can be obtained fromthe manufacturerclontech.com/techinfo/vectors/vectorsF-I/pdf/pIRES2-EGFPseq.pdf). Asimilar IRES can also be obtained from plasmid pCITE4a (Novagen, MadisonWis.; see also U.S. Pat. No. 4,937,190 which is herein incorporated byreference) by PCR using primers specific for the 5′ and 3′ ends of theCITE from nucleotides 16 to 518 in plasmid pCITE4a (the completesequence of pCITE4a is available atnovagen.com/docs/NDIS/69913-000.HTM). on plasmids pCITE4a-c (Novagen,URL:-novagen.com; U.S. Pat. No. 4,937,190 which is herein incorporatedby reference); pSLIRES11 (Accession: AF171227; pPV (Accession # Y07702);pSVIRES-N (Accession #: AJ000156); Creancier et al. J. Cell Biol., 10:275–281 (2000); Ramos and Martinez-Sala, RNA, 10: 1374–1383 (1999);Morgan et al. Nucleic Acids Res., 20: 1293–1299 (1992); Tsukiyama-Koharaet al. J. Virol., 66: 1476–1483 (1992); Jang and Wimmer et al. GenesDev., 4: 1560–1572 (1990)), or on the dicistronic retroviral vector(Accession #: D88622); or found in eukaryotic cells such as thefibroblast growth factor 2 IRES for stringent tissue-specific regulation(Creancier, et al., J. Cell. Biol., 150:275 (2000)) or theInternal-ribosome-entry-site of the 3′-untranslated region of the mRNAfor the beta subunit of mitochondrial H⁺-ATP synthase (Izquierdo andCuezva, Biochem. J., 346:849 (2000)).

Non-commercial sources of IRES's are also available and can be locatedas follows. Thus, plasmid pIRES-G (Hobbs, S. M. CRC Centre for CancerTherapeutics, Institute of Cancer Research, Block F, 15, Cotswold Road,Belmont, Sutton, Surrey SM2 5NG, UK) will serve as source of IRES andthe sequence of this plasmid is available (Genebank accession no.Y11034). Furthermore, an Internet search using the NCBI nucleotidedatabase ncbi.nlm.nih.gov/entrez/query.fcgi?CMD=Display&DB=nucleotide)and the search parameter “IRES not patent” yields 41 Files containingIRES sequences. Finally, IRES cDNA can be made synthetically using anApplied Biosystems ABI™ 3900 High-Throughput DNA Synthesizer (FosterCity, Calif. 94404 U.S.A.), using procedures provided by themanufacturer. To synthesize large IRES sequences such as the 502 bp IRESin pCITE4a, a series of segments are generated by PCR and ligatedtogether to form the full-length sequence using procedures well know inthe art [41–43]. Smaller IRES sequences such as the 53 bp IRES inhepatitis C virus (Genebank accession no. 1KH6_A; [45,46]) can be madesynthetically in a single round using an Applied Biosystems ABI™ 3900High-Throughput DNA Synthesizer (Foster City, Calif. 94404 U.S.A.) andprocedures provided by the manufacturer.

Examples of Genes of Interest that Can Be Inserted in dsRP

In the present invention, the gene of interest (GOI) introduced on aeukaryotic translation expression cassette into the rdsRP may encode animmunogen, which may be either a foreign immunogen from viral, bacterialand parasitic pathogens, or an endogenous immunogen, such as but notlimited to an autoimmune antigen or a tumor antigen. The immunogens maybe the full-length native protein, chimeric fusions between the foreignimmunogen and an endogenous protein or mimetic, a fragment or fragmentsthereof of an immunogen that originates from viral, bacterial andparasitic pathogens.

As used herein, “foreign immunogen” means a protein or fragment thereof,which is not normally expressed in the recipient animal cell or tissue,such as, but not limited to, viral proteins, bacterial proteins,parasite proteins, cytokines, chemokines, immunoregulatory agents, ortherapeutic agents.

An “endogenous immunogen” means a protein or part thereof that isnaturally present in the recipient animal cell or tissue, such as, butnot limited to, an endogenous cellular protein, an immunoregulatoryagent, or a therapeutic agent.

Alternatively or additionally, the immunogen may be encoded by asynthetic gene and may be constructed using conventional recombinant DNAmethods (See above).

The foreign immunogen can be any molecule that is expressed by anyviral, bacterial, or parasitic pathogen prior to or during entry into,colonization of, or replication in their animal host; the rdsRP mayexpress immunogens or parts thereof that originate from viral, bacterialand parasitic pathogens. These pathogens can be infectious in humans,domestic animals or wild animal hosts.

The viral pathogens, from which the viral antigens are derived, include,but are not limited to, Orthomyxoviruses, such as influenza virus(Taxonomy ID: 59771; Retroviruses, such as RSV, HTLV-1 (Taxonomy ID:39015), and HTLV-II (Taxonomy ID: 11909), Herpesviruses such as EBVTaxonomy ID: 10295); CMV (Taxonomy ID: 10358) or herpes simplex virus(ATCC #: VR-1487); Lentiviruses, such as HIV-1 (Taxonomy ID: 12721) andHIV-2 Taxonomy ID: 11709); Rhabdoviruses, such as rabies; Picomoviruses,such as Poliovirus (Taxonomy ID: 12080); Poxviruses, such as vaccinia(Taxonomy ID: 10245); Rotavirus (Taxonomy ID: 10912); and Parvoviruses,such as adeno-associated virus 1 (Taxonomy ID: 85106).

Examples of viral antigens can be found in the group including but notlimited to the human immunodeficiency virus antigens Nef (NationalInstitute of Allergy and Infectious Disease HIV Repository Cat. # 183;Genbank accession # AF238278), Gag, Env (National Institute of Allergyand Infectious Disease HIV Repository Cat. # 2433; Genbank accession #U39362), Tat (National Institute of Allergy and Infectious Disease HIVRepository Cat. # 827; Genbank accession # M13137), mutant derivativesof Tat, such as Tat-Δ31-45 (Agwale et al. Proc. Natl. Acad. Sci. Inpress. July 8^(th) (2002)), Rev (National Institute of Allergy andInfectious Disease HIV Repository Cat. # 2088; Genbank accession #L14572), and Pol (National Institute of Allergy and Infectious DiseaseHIV Repository Cat. # 238; Genbank accession # AJ237568) and T and Bcell epitopes of gp120 (Hanke and McMichael, AIDS Immunol Lett., 66:177(1999); Hanke, et al., Vaccine, 17:589 (1999); Palker et al, J.Immunol., 142:3612–3619 (1989)) chimeric derivatives of HIV-1 Env andgp120, such as but not restricted to fusion between gp120 and CD4 (Foutset al., J. Virol. 2000, 74:11427–11436 (2000)); truncated or modifiedderivatives of HIV-1 env, such as but not restricted to gp140 (Stamatoset al. J Virol, 72:9656–9667 (1998)) or derivatives of HIV-1 Env and/orgp140 thereof (Binley, et al. J Virol, 76:2606–2616 (2002); Sanders, etal. J Virol, 74:5091–5100 (2000); Binley, et al. J Virol, 74:627–643(2000)), the hepatitis B surface antigen (Genbank accession # AF043578;Wu et al, Proc. Natl. Acad. Sci., USA, 86:4726–4730 (1989)); rotavirusantigens, such as VP4 (Genbank accession # AJ293721; Mackow et al, Proc.Natl. Acad. Sci., USA, 87:518–522 (1990)) and VP7 (GenBank accession #AY003871; Green et al, J. Virol., 62:1819–1823 (1988)), influenza virusantigens such as hemagglutinin or (GenBank accession # AJ404627; Pertmerand Robinson, Virology, 257:406 (1999)); nucleoprotein (GenBankaccession # AJ289872; Lin et al, Proc. Natl. Acad. Sci., 97: 9654–9658(2000))) herpes simplex virus antigens such as thymidine kinase (Genbankaccession # AB047378; Whitley et al, In: New Generation Vaccines, pages825–854).

The bacterial pathogens, from which the bacterial antigens are derived,include but are not limited to, Mycobacterium spp. (tubercolis antigenswhich may be used in the practice of the present invention are describedin U.S. Pat. Nos. 5,955,077; 6,224,881; 6,384,018; 6,531,138; 6,596,218;U.S. published application 2002/0176867; and U.S. published application2003/0143243 each of which are herein incorporated by reference),Helicobacter pylori, Salmonella spp., Shigella spp., E. coli, Rickettsiaspp., Listeria spp., Legionella pneumoniae, Pseudomonas spp., Vibriospp., and Borellia burgdorferi.

Examples of protective antigens of bacterial pathogens include thesomatic antigens of enterotoxigenic E. coli, such as the CFA/I fimbrialantigen (Yamamoto et al., Infect. Immun., 50:925–928 (1985)) and thenontoxic B-subunit of the heat-labile toxin (Klipstein et al, Infect.Immun., 40:888–893 (1983)); pertactin of Bordetella pertussis (Robertset al, Vacc., 10:43–48 (1992)), adenylate cyclase-hemolysin of B.pertussis (Guiso et al, Micro. Path., 11:423–431 (1991)), fragment C oftetanus toxin of Clostridium tetani (Fairweather et al., Infect. Immun.,58:1323–1326 (1990)), OspA of Borellia burgdorferi (Sikand, et al.Pediatrics, 108:123–128 (2001); Wallich, et al. Infect Immun,69:2130–2136 (2001)), protective paracrystalline-surface-layer proteinsof Rickettsia prowazekii and Rickettsia typhi (Carl, et al. Proc NatlAcad Sci USA, 87:8237–8241 (1990)), the listeriolysin (also known as“Llo” and “Hly”) and/or the superoxide dismutase (also know as “SOD” and“p60”) of Listeria monocytogenes (Hess, J., et al. Infect. Immun.65:1286–92 (1997); Hess, J., et al. Proc. Natl. Acad. Sci. 93:1458–1463(1996); Bouwer, et al. J. Exp. Med. 175:1467–71 (1992)), the urease ofHelicobacter pylori (Gomez-Duarte, et al. Vaccine 16, 460–71 (1998);Corthesy-Theulaz, et al. Infection & Immunity 66, 581–6 (1998)), and thereceptor-binding domain of lethal toxin and/or the protective antigen ofBacillus anthrax (Price, et al. Infect. Immun. 69, 4509–4515 (2001)).

The parasitic pathogens, from which the parasitic antigens are derived,include but are not limited to, Plasmodium spp., such as Plasmodiumfalciparum (ATCC#: 30145); Trypanosome spp., such as Trypanosoma cruzi(ATCC#: 50797); Giardia spp., such as Giardia intestinalis (ATCC#:30888D); Boophilus spp., Babesia spp., such as Babesia microti (ATCC#:30221); Entamoeba spp., such as Entamoeba histolytica (ATCC#: 30015);Eimeria spp., such as Eimeria maxima (ATCC# 40357); Leishmania spp.(Taxonomy ID: 38568); Schistosome spp., Brugia spp., Fascida spp.,Dirofilaria spp., Wuchereria spp., and Onchocerea spp.

Examples of protective antigens of parasitic pathogens include thecircumsporozoite antigens of Plasmodium spp. (Sadoff et al, Science,240:336–337 (1988)), such as the circumsporozoite antigen of P. bergeriior the circumsporozoite antigen of P. falciparum; the merozoite surfaceantigen of Plasmodium spp. (Spetzler et al, Int. J. Pept. Prot. Res.,43:351–358 (1994)); the galactose specific lectin of Entamoebahistolytica (Mann et al, Proc. Natl. Acad. Sci., USA, 88:3248–3252(1991)), gp63 of Leishmania spp. (Russell et al, J. Immunol.,140:1274–1278 (1988); Xu and Liew, Immunol., 84: 173–176 (1995)), gp46of Leishmania major (Handman et al, Vaccine, 18: 3011–3017 (2000)paramyosin of Brugia malayi (Li et al, Mol. Biochem. Parasitol.,49:315–323 (1991)), the triose-phosphate isomerase of Schistosomamansoni (Shoemaker et al, Proc. Natl. Acad. Sci., USA, 89:1842–1846(1992)); the secreted globin-like protein of Trichostrongyluscolubriformis (Frenkel et al, Mol. Biochem. Parasitol., 50:27–36(1992)); the glutathione-S-transferase's of Frasciola hepatica (Hillyeret al, Exp. Parasitol., 75:176–186 (1992)), Schistosoma bovis and S.japonicum (Bashir et al, Trop. Geog. Med., 46:255–258 (1994)); and KLHof Schistosoma bovis and S. japonicum (Bashir et al, supra).

As mentioned earlier, the dsRP vaccine may encode an endogenousimmunogen, which may be any cellular protein, immunoregulatory agent, ortherapeutic agent, or parts thereof, that may be expressed in therecipient cell, including but not limited to tumor, transplantation, andautoimmune immunogens, or fragments and derivatives of tumor,transplantation, and autoimmune immunogens thereof. Thus, in the presentinvention, dsRP may encode tumor, transplant, or autoimmune immunogens,or parts or derivatives thereof. Alternatively, the dsRP may encodesynthetic genes (made as described above), which encode tumor-specific,transplant, or autoimmune antigens or parts thereof.

Examples of tumor specific antigens include prostate specific antigen(Gattuso et al, Human Pathol., 26:123–126 (1995)), TAG-72 and CEA(Guadagni et al, Int. J. Biol. Markers, 9:53–60 (1994)), MAGE-1 andtyrosinase (Coulie et al, J. Immunothera., 14:104–109 (1993)). Recentlyit has been shown in mice that immunization with non-malignant cellsexpressing a tumor antigen provides a vaccine effect, and also helps theanimal mount an immune response to clear malignant tumor cellsdisplaying the same antigen (Koeppen et al, Anal. N.Y. Acad. Sci.,690:244–255 (1993)).

Examples of transplant antigens include the CD3 molecule on T cells(Alegre et al, Digest. Dis. Sci., 40:58–64 (1995)). Treatment with anantibody to CD3 receptor has been shown to rapidly clear circulating Tcells and reverse cell-mediated transplant rejection (Alegre et al,supra).

Examples of autoimmune antigens include IAS β chain (Topham et al, Proc.Natl. Acad. Sci., USA, 91:8005–8009 (1994)). Vaccination of mice with an18 amino acid peptide from IAS β chain has been demonstrated to provideprotection and treatment to mice with experimental autoimmuneencephalomyelitis (Topham et al, supra).

Introduction of Sequences into dsRP

To manipulate dsRP, cDNA copies of the mRNA segments L, M and S aregenerated and inserted into a prokaryotic expression vector (FIG. 2)using procedures well know in the art (Ausubel et al, supra; andSambrook, supra). These cloned cDNA copies of the mRNA are used astarget sequences into which the sequence of interest that encodes theGOI is inserted (FIGS. 3 and 4).

To generate rdsRP that retain the capacity to produce infectious phage,the sequence that is being incorporated into the dsRP can be insertedinto an unessential region of a dsRP, such as but not limited to the PstI restriction endonuclease site in the cDNA clone of M segment [17].Alternatively, standard PCR techniques can be used to introduce REdigestion sites in a non-essential region, such as between the pacsequence in segment-M and gene-10 in Phi-6 [17].

Alternatively, the sequence that is being incorporated into the dsRP canreplace genes of the dsRP that are not required for the production ofstable nucleocapsids, such as but not limited to the replacement ofgene-10 in segment-M, gene-3 in segment-M, gene-9 in segment-S, gene-12in segment-S; alternatively the sequence being inserted into the dsRP.Thus, plasmid pLM656 (From Dr. L Mindich, Department of Microbiology,The Public Health Research Institute NY, N.Y.; [17]), carries thecomplete cDNA copy of segment-M, is digested with RE Pst I and theresultant linear plasmid DNA is treated with T4 DNA polymerase to removethe single stranded sequences created by Pst I thereby creatingblunt-ends. Sequences of interest can be inserted into Pst I-digested,T4 polymerase-treated pLM656 DNA by standard blunt-end ligationtechniques using T4 DNA ligase (Ausubel et al, supra; and Sambrook,supra). The resultant plasmid carries a cDNA copy of the recombinantsegment M produce mRNA's that carry the sequence of interest.

Introduction of Functional Eukaryotic Translation Expression Cassettesinto dsRP

As indicated above, in one embodiment of the current invention,sequences of interest can encode a functional eukaryotic translationexpression cassette. A simple approach to obtain a functional eukaryotictranslation expression cassette is to introduce an IRES functionallylinked to a gene of interest (herein referred to as GOI), which isnormally placed downstream (i.e. 3′) of the IRES.

Sequences encoding the IRES can be amplified by PCR using primersspecific for the 5′ and 3′ ends of the IRES sequence; the GOI can beamplified using primers specific for the 5′ and 3′ ends of thetranscribed region of the GOI or parts thereof. RE digestion sites (e.g.Not I, Eco RI, Sal I) can be introduced into the primers so that theresultant PCR-generated products can be digested with said REs and fusedto a positive-selection allele (herein referred to as “PSA”), which canbe amplified using PCR primers that place RE recognition sites (e.g. NotI) at the 5′ and 3′ ends of the PSA. The particular PSA used in thecurrent invention is not critical thereto and can be the kan^(r) allelein plasmid pUC18K1 [47]; the Escherichia coli asd allele in plasmidpYA292 (Galan, et al., Gene 94:29–35 (1990); Genbank accession no.V00262).

The resultant chimeric fragment encoding PSA::IRES::GOI is inserted intoan RE-digested plasmid containing the target dsRP segment (e.g.insertion into the M-segment using Pst I-digested, T4 polymerase-treatedpLM656 DNA and blunt-end ligation to the PSA::IRES::GOI sequence, asabove (Ausubel et al, supra; and Sambrook, supra). The resultantplasmid, carries a cDNA copy of the recombinant segment and producesmRNA's that bear a PSA for maintenance of the recombinant segment in therdsRP, a cap-independent translation recognition sequence (i.e. IRES)and an GOI reporter gene.

Generation of rdsRP

An application of the current invention entails the use of enriched orpurified rdsRP for direct administration to eukaryotic cells or tissues.The particular dsRP is not critical to the present invention andincludes but is not restricted to one of Phi-6 (Genbank accession no.M17461 (Segment-L), M17462 (Segment-M), M12921 (Segment-S)); Phi-8(Genbank accession no. AF226851 (Segment-L), NC_(—)003300 (Segment-M),AF226853 (Segment-S)); and Phi-13 (Genbank accession no. AF261668(Segment-L), AF261668 (Segment-M), NC_(—)003714 (Segment-S)) and areavailable from Dr. L. Mindich at Department of Microbiology, PublicHealth Research Institute, NY, N.Y.

DsRP phi-6 normally replicates in Pseudomonas syringae [5]; dsRP phi-8,phi-11, phi-12 and phi-13 replicate in Escherichia coli strain JM109(American type tissue culture collection (herein “ATCC”) # 53323) andO-antigen negative mutants of Salmonella enterica serovar Typhimurium(herein designated “S. typhimurium”) [5,14–16].

Alternatively, the cDNA sequences encoding the dsRP can be generatedsynthetically using an Applied Biosystems ABI™ 3900 High-Throughput DNASynthesizer (Foster City, Calif. 94404 U.S.A.) and procedures providedby the manufacturer. To synthesize the cDNA copies of segments L, M andS a series of segments of the full-length sequence are generated by PCRand ligated together to form the full-length segment using procedureswell know in the art [41–43]. Briefly, synthetic oligonucleotides100–200 nucleotides in length (i.e. preferably with sequences at the 5′-and 3′ ends that match at the 5′ and 3′ ends of the oligonucleotidesthat encodes the adjacent sequence) are produced using an automated DNAsynthesizer (E.g. Applied Biosystems ABI™ 3900 High-Throughput DNASynthesizer (Foster City, Calif. 94404 U.S.A.)). Using the sameapproach, the complement oligonucleotides are synthesized and annealedwith the complementary partners to form double strandedoligonucleotides. Pairs of double stranded oligonucleotides (i.e. thosethat encode adjacent sequences) and joined by ligation to form a largerfragment. These larger fragments are purified by agarose gelelectrophoresis and isolated using a gel purification kit (E.g. TheQIAEX® II Gel Extraction System, from Qiagen, Santa Cruz, Calif., Cat.No. 12385). This procedure is repeated until the full-length DNAmolecule is created. After each round of ligation the fragments can beamplified by PCR to increase the yield. Procedures for de novo syntheticgene construction are well known in the art, and are described elsewhere(Andre et al., supra, (1998); et al., Haas supra, (1996)); alternativelysynthetic genes can be purchased commercially, e.g. from the MidlandCertified Reagent Co. (Midland, Tex.).

To genetically manipulate dsRP, phage are amplified in a bacterial hoststrain, including but not limited to Pseudomonas syringae [5],Escherichia coli strain JM109 (American type tissue culture collection(herein “ATCC”) # 53323) and O-antigen negative mutants of Salmonellaenterica serovar Typhimurium (herein designated “S. typhimurium”)[5,14–16]. and purified to the level commensurate to the desired invitro or in vivo application. RdsRP are produced from strains carryingthe plasmids that encode the cDNA copies of the manipulated segments(i.e. the segments containing the inserted sequences of interestencoding the functional eukaryotic translation expression cassette,catalytic RNA or antisense RNA).

To produce rdsRP in Escherichia coli (e.g. strain JM109), thecDNA-containing plasmids (e.g. pLM656-PSA::IRES::EGFP) are introducedinto target bacterial strains by standard bacterial transformationmethods (Ausubel et al, supra; and Sambrook, supra) andantibiotic-resistant transformants are isolated in solid media (e.g.Luria-Bertani agar (herein referred to as LBA), Difco Detroit Mich.)containing the appropriate antibiotic (Antibiotics for inclusion inbacteriological media are available from Sigma, St. Louis Mo.) at aconcentration that is equal to, or greater than, the minimal inhibitoryconcentration (also referred to as the “mic”).

The bacterial isolates are cultured at temperatures that range from 25°C. to 44° C. for 16 to 48 hr; however, it is preferable to culture thetransformants at 37° C. for 16 hr. Colonies that grow on the selectivesolid media are subsequently isolated and purified by standard methods(Ausubel et al, supra; and Sambrook, supra). To verify that theantibiotic-resistant isolates carrying the plasmid of interest,individual isolates are cultured in liquid media (e.g. Luria-Bertanibroth (herein referred to as “LB”), Difco Mo.). The transformants areharvested after cultures reach an optical density at 600 nm (OD₆₀₀) of0.001 to 4.0, preferably 0.9, relative to the OD₆₀₀ a sterile LBcontrol. Plasmid DNA is isolated from these cultures and analysed by REdigestion using enzymes that generate a defined digestion pattern basedon the predicted sequence of the recombinant plasmid, including but notlimited to Eco RI, Pst I, Hind III, Hae I, Sau IIIa, Not I, and Sal I.Alternatively or in addition, the plasmids can be screened by PCR usingprimers that amplify defined fragments within the recombinant plasmid,including but not limited to PCR primers that amplify the dsRP segment,the PSA, the IRES and the GOI. The PCR primers for the amplificationsare designed using Clone Manager® software version 4.1 (Scientific andEducational Software Inc., Durhan N.C.). This software enables thedesign PCR primers and identifies RE sites that are compatible with thespecific DNA fragments being manipulated. PCRs are conducted in aStrategene Robocycler, model 400880 (Strategene) and primer annealing,elongation and denaturation times in the PCRs are set according tostandard procedures (Ausubel et al, supra). The RE digestions and thePCRs are subsequently analyzed by agarose gel electrophoresis usingstandard procedures (Ausubel et al, supra; and Sambrook, supra). Apositive clone is defined as one that displays the appropriate REpattern and/or PCR pattern. Plasmids identified through this procedurecan be further evaluated using standard DNA sequencing procedures, asdescribed above.

Having identified the desired transformants, individual strains arestored in a storage media, which is LB containing 50% (v/v) glycerol;Bacterial isolates are harvested from solid media using a sterile cottonwool swab and suspended in storage media at a density of 10⁹ cfu/ml andthe suspensions are stored at −80° C.

Isolation and Purification of rdsRP

Batches of rdsRP are generated by replicating a parent rdsRP in thebacterial transformant said expresses the recombinant segment (FIG. 4).Methods for incorporation of recombinant segments into dsRP and for thesubsequent replication, isolation and purification of the resultantrdsRP are well known in the art and have been published extensively indetail elsewhere (Mindich, et al. J Virol 66, 2605–10 (1992); Mindich,et al. Virology 212:213–217 (1995); Mindich, et al., J Bacteriol181:4505–4508 (1999); Qiao, et al., Virology 275:218–224 (2000); Qiao,et al., Virology 227:103–110 (1997); Olkkonen, et al., Proc Natl AcadSci USA 87:9173–9177 (1990); Onodera, et al., J Virol 66, 190–196(1992)).

Development of rdsRP that Express an Adjuvant

Recombinant dsRP can be constructed that encode an immunogen and anadjuvant, and can be used to increase host responses to the dsRP.Alternatively, recombinant dsRP can be constructed that encode anadjuvant, in mixtures with other dsRP to increase host responses toimmunogens encoded by the partner rdsRP.

The particular adjuvant encoded by the rdsRP is not critical to thepresent invention and may be the A subunit of cholera toxin (i.e. CtxA;GenBank accession no. X00171, AF175708, D30053, D30052), or partsthereof (E.g. the A1 domain of the A subunit of Ctx (i.e. CtxA1; GenBankaccession no. K02679)), from any classical Vibrio cholerae (E.g. V.cholerae strain 395, ATCC # 39541) or El Tor V. cholerae (E.g. V.cholerae strain 2125, ATCC # 39050) strain. Alternatively, any bacterialtoxin that increases cellular cAMP levels, such as a member of thefamily of bacterial adenosine diphosphate-ribosylating exotoxins(Krueger and Barbier, Clin. Microbiol. Rev., 8:34 (1995)), may be usedin place of CtxA, for example the A subunit of heat-labile toxin(referred to herein as EltA) of enterotoxigenic Escherichia coli(GenBank accession # M35581), pertussis toxin S1 subunit (E.g. ptxS1,GenBank accession # AJ007364, AJ007363, AJ006159, AJ006157, etc.); as afurther alternative the adjuvant may be one of the adenylatecyclase-hemolysins of Bordetella pertussis (ATCC # 8467), Bordetellabronchiseptica (ATCC # 7773) or Bordetella parapertussis (ATCC # 15237),E.g. the cyaA genes of B. pertussis (GenBank accession no. X14199), B.parapertussis (GenBank accession no. AJ249835) or B. bronchiseptica(GenBank accession no. Z37112).

Alternatively, the particular the adjuvant may be devoid ofADP-ribosyltransferase activity and may be any derivative of the Asubunit of cholera toxin (i.e. CtxA; GenBank accession no. X00171,AF175708, D30053, D30052,), or parts thereof (i.e. the A1 domain of theA subunit of Ctx (i.e. CtxA1; GenBank accession no. K02679)), from anyclassical Vibrio cholerae (E.g. V. cholerae strain 395, ATCC # 39541) orEl Tor V. cholerae (E.g. V. cholerae strain 2125, ATCC # 39050) thatlack ADP-ribosyltransferase catalytic activity but retain the structuralintegrity, including but not restricted to replacement of arginine-7with lysine (herein referred to as “R7K”), serine-61 with lysine (S61K),serine-63 with lysine (S63K), valine-53 with aspartic acid (V53D),valine-97 with lysine (V97K) or tyrosine-104 with lysine (Y104K), orcombinations thereof. Alternatively, the particularADP-ribosyltransferase toxin that is devoid of ADP-ribosyltransferaseactivity may be any derivative of cholera toxin that fully assemble, butare nontoxic proteins due to mutations in the catalytic-site, oradjacent to the catalytic site, respectively. Such mutants are made byconventional site-directed mutagenesis procedures, as described above.

As a further alternative, the adjuvant of ADP-ribosyltransferaseactivity may be any derivative of the A subunit of heat-labile toxin(referred to herein as “LTA” of enterotoxigenic Escherichia coli(GenBank accession # M35581) isolated from any enterotoxigenicEscherichia coli, including but not restricted to E. coli strain H10407(ATCC # 35401) that lack ADP-ribosyltransferase catalytic activity butretain the structural integrity, including but not restricted to R7K,S61K, S63K, V53D, V97K or Y104K, or combinations thereof. Alternatively,the particular ADP-ribosyltransferase toxin that is devoid ofADP-ribosyltransferase activity may be any derivative of cholera toxinthat fully assemble, but are nontoxic proteins due to mutations in thecatalytic-site, or adjacent to the catalytic site, respectively. Suchmutants are made by conventional site-directed mutagenesis procedures,as described above.

Development of dsRP that Express an Immunoregulatory Agent

Recombinant dsRP can be constructed that encode an immunogen and acytokine, and can be used to increase host responses to the dsRP.Alternatively, recombinant dsRP can be constructed that encode saidcytokine alone, in mixtures with other dsRP to increase host responsesto immunogens encoded by the partner rdsRP.

The particular cytokine encoded by the rdsRP is not critical to thepresent invention includes, but not limited to, interleukin-4 (hereinreferred to as “IL-4”; Genbank accession no. AF352783 (Murine IL-4) orNM_(—)000589 (Human IL-4)), IL-5 (Genbank accession no. NM_(—)010558(Murine IL-5) or NM_(—)000879 (Human IL-5)), IL-6 (Genbank accession no.M20572 (Murine IL-6) or M29150 (Human IL-6)), IL-10 (Genbank accessionno. NM_(—)010548 (Murine IL-10) or AF418271 (Human IL-10)), I1-12_(p40)(Genbank accession no. NM_(—)008352 (Murine IL-12 p40) or AY008847(Human IL-12 p40)), IL-12_(p70) (Genbank accession no.NM_(—)008351/NM_(—)008352 (Murine IL-12 p35/40) or AF093065/AY008847(Human IL-12 p35/40)), TGFβ (Genbank accession no. NM_(—)011577 (MurineTGFβ1) or M60316 (Human TGFβ1)), and TNFα Genbank accession no. X02611(Murine TNFα) or M26331 (Human TNFα)).

Recombinant DNA and RNA procedures for the introduction of functionaleukaryotic translation expression cassettes to generate rdsRP capable ofexpressing an immunoregulatory agent in eukaryotic cells or tissues aredescribed above, wherein said immunoregulatory agent is the GOI.

Development of Self-amplifying dsRP

RdsRP can be constructed that carry an alpha-virus self-amplifyingexpression system (Pushko, et al., Virology 239:389–401 (1997); Caley,et al. J Virol 71:3031–3038 (1997); Mossman, et al., J Virol 70,1953–1960 (1996); Zhou, et al., Vaccine 12:1510–1514 (1994)) and areused to significantly elevate the expression of the GOI. The particularalpha-virus self-amplifying expression system is not critical to thepresent invention and can be selected from semliki forest virus, such asbut not limited to the semliki forest virus replicon in commerciallyavailable plasmid pSFV1 from Invitrogen Inc., or sequences encoding thenonstructural protein precursor and replicase recognition sequences ofVenezuela equine encephalitis virus (i.e Genbank accession no. L04653).

Recombinant DNA, PCR, RE and sequence analysis procedures for theintroduction of functional eukaryotic translation expression cassettesinto rdsRP that incorporates an alpha-virus self-amplifying expressionsystem capable functionally linked to an immunogen, immunoregulatoryagent, or therapeutic agent, are described above, wherein saidimmunoregulatory agent constitutes part of the GOI and the immunogen,immunoregulatory agent or therapeutic agent are placed downstream of thereplicase recognition sequence (Genbank accession no. L04653), asdescribed (Pushko, et al., Virology 239:389–401 (1997); Caley, et al. JVirol 71:3031–3038 (1997); Mossman, et al., J Virol 70, 1953–1960(1996); Zhou, et al., Vaccine 12:1510–1514 (1994)).

Administration of rdRP to Dendritic Cells In Vitro

The present invention can be used in vaccination regimens, wherein humanderived dendritic cells are pulsed with the rdsRP and subsequentlyinjected into an animal, intravenously, subcutaneously orintramuscularly. Such in vitro vaccination protocols are useful for theinduction of anti-tumor immune responses.

Methods for the production and culture of dendritic cells are well knownin the art and described elsewhere (Sallusto et al. 1994)). In short,human PBMCs are separated from the blood of healthy donors bycentrifugation in Histopaque 1077 (Sigma, St. Louis, Mo.). The cells areenriched for monocytes (90–95% pure) using the StemSep MonocyteEnrichment Cocktail and a magnetic negative-selection column protocol(StemSep, Vancouver, British Columbia). Following enrichment, themonocytes are plated in RPMI 1640 (Gibco BRL: Grand Island, N.Y.) andincubated for 2 hours at 37° C. in a 5% CO₂ (37° C./5% CO₂).Non-adherent cells and media are removed and replaced with complete DCmedia, which comprises of RPMI 1640 supplemented with 10% fetal bovineserum (Gibco-BRL), 1% sodium pyruvate (Sigma), 1% non-essential aminoacids (Gibco-BRL), Gentamycin (Gibco-BRL), 50 μM β-mecaptoethanal(Sigma), 10 μM Hepes (Sigma), 35 ng/ml interleukin-4 (IL-4, R+D Systems,Minnesota, Minn.), and 50 ng/ml granulocyte/monocyte-colony stimulatingfactor (GM-CSF, R+D Systems). Cells develop the appearance and cellsurface phenotype of immature MDDCs after 4 days in culture at 37° C. 5%CO₂ environment, as confirmed by microscopy.

The DCs are analysed by flow cytometry at various times during theprocedure to ensure that the appropriate antigen presenting propertiesare activated. The DCs are harvested in phosphate buffered saline(Gibco-BRL) supplemented with 2% human AB serum (Sigma) and 0.1% azide(Sigma), stained with R-phycoerythrin (PE)-anti-CD80, FITC-anti-CD83,PE-anti-CD86, PE-anti-CD25, PE-anti-HLA-ABC, and PE-anti-HLA-DR, (BectonDickerson Pharmingen: San Deigo, Calif.) and fixed in 2%paraformaldehyde (Sigma) in PBS. Single-label flow cytometry data arecollected using a FACSCaliber® (Beckmon Dickerson); expression ofmaturation markers in large cells is analyzed using CellQuest® (BeckmonDickerson) and FlowJo® software (TreeStar, Stanford, Calif.).

To assess the cytokines produced by the DCs, Semi-quantitative ELISAassays for IL-6, TNF-α, IL-10, IL-12 p40, and IL-12 p70 (R+D Systems)were performed according to manufacturers instructions. In thoseexperiments where cell surface data was not acquired, the cells andsupernatants were frozen at −20° C. in the wells in which they wereplated. The cells and supernatants were thawed and spun at 2000 RPM for15 minutes to remove particulate matter immediately before ELISA assayswere performed. In other experiments, the cell supernatants werereserved and either incorporated immediately into the ELISA protocol orfrozen at −20° C.

Formulation of rdsRP Vaccines for In Vivo Administration

The specific method used to formulate the novel rdsRP vaccines describedherein is not critical to the present invention and can be selected froma physiological buffer (Felgner et al., U.S. Pat. No. 5,589,466 (1996));aluminum phosphate or aluminum hydroxyphosphate (e.g. Ulmer et al.,Vaccine, 18:18 (2000)), monophosphoryl-lipid A (also referred to as MPLor MPLA; Schneerson et al. J. Immunol., 147: 2136–2140 (1991); e.g.Sasaki et al. Inf. Immunol., 65: 3520–3528 (1997); Lodmell et al.Vaccine, 18: 1059–1066 (2000)), QS-21 saponin (e.g. Sasaki, et al., J.Virol., 72:4931 (1998); dexamethasone (e.g. Malone, et al., J. Biol.Chem. 269:29903 (1994); CpG DNA sequences (Davis et al., J. Immunol.,15:870 (1998); or lipopolysaccharide (LPS) antagonist (Hone et al.,supra (1997)).

Administration of rdsRP

The rdsRP vaccine can be administered directly into animal tissues byintravenous, intramuscular, intradermal, intraperitoneally, intranasaland oral inoculation routes. The specific method used to introduce therdsRP vaccines described herein into the target animal tissue is notcritical to the present invention and can be selected from previouslydescribed vaccination procedures (Wolff, et al., Biotechniques 11:474–85(1991); Johnston and Tang, Methods Cell Biol 43:353–365 (1994); Yang andSun, Nat Med 1:481–483 (1995); Qiu, et al., Gene Ther. 3:262–8 (1996);Larsen, et al., J. Virol. 72:1704–8 (1998); Shata and Hone J. Virol.75:9665–9670 (2001); Shata, et al., Vaccine 20:623–629 (2001); Ogra, etal., J Virol 71:3031–3038 (1997); Buge, et al., J. Virol. 71:8531–8541(1997);Belyakov, et al., Nat. Med. 7, 1320–1326 (2001); Lambert, et al.,Vaccine 19:3033–3042 (2001); Kaneko, et al. Virology 267: 8–16 (2000);Belyakov, et al., Proc Natl Acad Sci USA 96:4512–4517 (1999).

Oral Administration of rdsRP with Bacterial Vaccine Vectors

Oral vaccination of the target animal with the rdsRP of the presentinvention can also be achieved using a non-pathogenic or attenuatedbacterial vaccine vector. The amount of the bacterial vaccine vector tobe administered with the rdsRP of the present invention will varydepending on the species of the subject, as well as the disease orcondition that is being treated. Generally, the dosage employed will beabout 10³ to 10¹¹ viable organisms, preferably about 10⁵ to 10⁹ viableorganisms.

The bacterial DNA vaccine vector and the rdsRP are generallyadministered along with a pharmaceutically acceptable carrier ordiluent. The particular pharmaceutically acceptable carrier or diluentemployed is not critical to the present invention. Examples of diluentsinclude a phosphate buffered saline, buffer for buffering againstgastric acid in the stomach, such as citrate buffer (pH 7.0) containingsucrose, bicarbonate buffer (pH 7.0) alone (Levine et al, J. Clin.Invest., 79:888–902 (1987); and Black et al J. Infect. Dis.,155:1260–1265 (1987)), or bicarbonate buffer (pH 7.0) containingascorbic acid, lactose, and optionally aspartame (Levine et al, Lancet,II:467–470 (1988)). Examples of carriers include proteins, e.g., asfound in skim milk, sugars, e.g., sucrose, or polyvinylpyrrolidone.Typically these carriers would be used at a concentration of about0.1–90% (w/v) but preferably at a range of 1–10% (w/v).

EXAMPLE 1 Recombinant DNA Procedures

Restriction endonucleases (herein “Res”); New England Biolabs Beverly,Mass.), T4 DNA ligase (New England Biolabs, Beverly, Mass.) and Taqpolymerase (Life technologies, Gaithersburg, Md.) were used according tothe manufacturers' protocols; Plasmid DNA was prepared using small-scale(Qiagen Miniprep^(R) kit, Santa Clarita, Calif.) or large-scale (QiagenMaxiprep^(R) kit, Santa Clarita, Calif.) plasmids DNA purification kitsaccording to the manufacturer's protocols (Qiagen, Santa Clarita,Calif.); Nuclease-free, molecular biology grade milli-Q water, Tris-HCl(pH 7.5), EDTA pH 8.0, 1M MgCl₂, 100% (v/v) ethanol, ultra-pure agarose,and agarose gel electrophoresis buffer were purchased from Lifetechnologies, Gaithersburg, Md. RE digestions, PCRs, DNA ligationreactions and agarose gel electrophoresis were conducted according towell-known procedures (Sambrook, et al., supra (1989); (Ausubel, et al.,supra (1990)).

Nucleotide sequencing to verify the DNA sequence of each recombinantplasmid described in the following examples was accomplished byconventional automated DNA sequencing techniques using an AppliedBiosystems automated sequencer, model 373A.

PCR primers were purchased from the University of Maryland BiopolymerFacility (Baltimore, Md.) and were synthesized using an AppliedBiosystems DNA synthesizer (model 373A). PCR primers were used at aconcentration of 200 μM and annealing temperatures for the PCR reactionswere determined using Clone manager software version 4.1 (Scientific andEducational Software Inc., Durhan N.C.). PCRs were conducted in aStrategene Robocycler, model 400880 (Strategene, La Jolla, Calif.). ThePCR primers for the amplifications are designed using Clone Manager®software version 4.1 (Scientific and Educational Software Inc., DurhanN.C.). This software enabled the design PCR primers and identifies REsites that were compatible with the specific DNA fragments beingmanipulated. PCRs were conducted in a Strategene Robocycler, model400880 (Strategene) and primer annealing, elongation and denaturationtimes in the PCRs were set according to standard procedures (Ausubel etal, supra). The RE digestions and the PCRs were subsequently analyzed byagarose gel electrophoresis using standard procedures (Ausubel et al,supra; and Sambrook, supra). A positive clone is defined as one thatdisplays the appropriate RE pattern and/or PCR pattern. Plasmidsidentified through this procedure can be further evaluated usingstandard DNA sequencing procedures, as described above.

Escherichia coli strain Sable2^(R) was purchased from Life Technologies(Bethesda, Md.) and served as initial host of the recombinant plasmidsdescribed in the examples below. Recombinant plasmids were introducedinto E. coli strain Stable2^(R) by electroporation using a Gene Pulser(BioRad Laboratories, Hercules, Calif.) set at 200Ω, 25 μF and 2.5 kV,as described (Ausubel et al, supra).

Bacterial strains were grown on tryptic soy agar (Difco, Detroit Mich.)or in tryptic soy broth (Difco, Detroit Mich.), which were madeaccording to the manufacturer's directions. Unless stated otherwise, allbacteria were grown at 37° C. When appropriate, the media weresupplemented with 100-μg/ml ampicillin (Sigma, St. Louis, Mo.).Bacterial strains were stored at −80° C. suspended in tryptic soy broth(Difco) containing 30% (v/v) glycerol (v/v; Sigma, St Louis Mo.) at ca.10⁹ colony-forming units (herein referred to as “cfu”) per ml.

EXAMPLE 2 Construction of a Prototype HIV-1 gp120 rdsRP Nucleocapsid

A functional eukaryotic translation expression cassette is obtained byincorporating an IRES that is functionally linked to the immunogen, thelatter being placed immediately downstream of the IRES. Expressionvector, designated “prφ8Seg-S”, carries the φ-8 segment-S pac sequenceand gene-8, a positive selection allele, the encephalomyocarditis virusIRES [48], multiple cloning sites, a poly-adenylation sequence and φ-8segment-S 3′-prime RNA-dependent RNA polymerase recognition sequence, asshown (FIG. 5). The blunt-end MscI site serves as an insertion point forany desired gene, such as those outlined in the detailed description ofthis invention above. Note that genes 5, 9 and 12 are omit in theresultant rdsRP, as these genes are not required for nucleocapsidproduction [15,20]. In addition, φ-8 segment-M is not utilizes, as it isnot required for nucleocapsid production and maintenance [15,20].

The components of plasmid prφ8Seg-S are assembled by joining thesequences obtained from the following sources:

-   -   1. The φ-8 segment-S pac sequence and gene-8 ([15]; Genbank        accession # AF226853) are obtained by PCR from plasmid pLM2755        (kindly provided by Dr. Leonard Mindich, Department of        Microbiology, Public Health Research Institute, NY, N.Y.).    -   2. A PSA encoding the Escherichia coli asd allele (Genbank        accession no. V00262) for maintenance of the recombinant        segment-S in the resultant rdsRP during propagation in        Escherichia coli [15] is obtained by PCR from plasmid pYA292        [49].    -   3. The encephalomyocarditis virus IRES is obtained from pCITE4a        by PCR, as described [50,51]. The 3-prime primer for this        amplification encodes oning sites including MscI, EcoRI, SalI        and NotI restriction endonuclease (RE) sites 3-prime to the IRES        sequence (MscI is a blunt-end RE and provides an ATG start codon        that is functionally linked to the IRES) and the bovine        poly-adenylation sequence (obtained from pcDNA3.1 (Invitrogen)).    -   4. Similarly, the φ-8 segment-S RNA-dependent RNA polymerase        recognition sequence is amplified from pLM2755 [15] by PCR.

The rdsRP is assembled using a sequential assembly procedure similar tothe procedure used to assemble synthetic genes [52]. Thus, PCR-generatedφ-8 segment-S pac sequence and gene-8 fragment is joined by T4 DNAligase to the PCR-generated E. coli asd allele. This fusion fragment isamplified by PCR using primers specific for the 5-prime and 3-primeends. Similarly, the PCR-generated encephalomyocarditis virus IRES::REsites::poly-A fragment is joined by T4 DNA ligase to the PCR-generatedφ-8 segment-S RNA-dependent RNA polymerase recognition sequence and theresultant fusion fragment is amplified by PCR using primers specific forthe 5-prime and 3-prime ends of the fusion fragment. The two fusionfragments are then joined by ligation and amplified by PCR as above.This fragment is then inserted into the SmaI site in broad host rangeexpression vector pBAD (Invitrogen, Carlsbad Calif.), which places theexpression of the recombinant segment-S under the tight control of theL-arabinose-inducible E. coli araBAD promoter (P_(BAD)). The resultantplasmid, designated “prφ8Seg-S” is isolated and purified as described inExample 1.

An rdsRP capable of expressing HIV-1 gp120 in mammalian cells isconstructed as follows. The sequence encoding syngp120 is obtained frompOGL1 by PCR so that MscI and NotI sites are created at the 5-prime and3-prime ends of syngp120, respectively, as before [39]. ThePCR-generated MscI::syngp120::NotI fragment is digested with MscI (NewEngland Biolabs) and NotI (New England Biolabs) and inserted using T4DNA ligase (New England Biolabs) into MscI-, NotI-digested prφ8Seg-S, asshown (FIG. 5); this procedure functionally links syngp120 to the IRES.The resultant plasmid is designated prdsRP-1 and rdsRP that incorporatethe recombinant segment-S expressed by prdsRP-1 (Example 7) bear thecapacity to express gp120 in mammalians cells.

EXAMPLE 3 Construction of a rdsRP that Expresses a ConformationallyConstrained HIV-1 Envelope Immunogen and Induces Broadly NeutralizingAntibodies to HIV-1

The advent of conformationally constrained HIV-1 envelope (Env)immunogens (i.e gp120-CD4 fusions herein referred to as “FLSC” [53] thatinduce antibodies capable of neutralizing a broad cross-section ofprimary HIV-1 isolates made it feasible to develop HIV-1 vaccinationstrategies that afford protection through humoral mechanisms. Therefore,a second-generation rdsRP vector is constructed by inserting sequencesencoding FLSC [53] in place of syngp120 using procedures described inexamples 1 and 2; the resultant rdsRP is designated “rdsRP-FLSC”.

It is important to note that there is direct evidence linking humoralimmune mechanisms to the prevention and control of HIV-1. In particular,data demonstrating that monoclonal and polyclonal neutralizingantibodies against HIV-1 or SIV transfer protection against homologouschallenge in animal models established direct evidence for protectionthrough a humoral mechanism [54–65]. Nevertheless, reports describingthe tertiary models of gp120 suggest that conserved epitopes exposedafter binding to CD4, which are pivotal targets of broadly neutralizingantibodies, lie concealed within the core structure of unbound gp120. Asa result, these key epitopes are poorly immunogenic in conventional Env,gp140 and gp120 subunit vaccines, which induce antibodies primarily tosurface-exposed epitopes [66–72]. However, CD4-bound, conformationallyconstrained gp120 immunogens, such as FLSC [66–70] expose crypticepitopes in gp120 that are normally only exposed following viralattachment to CD4 [66–70]. The availability of chemically andgenetically stabilized conformationally constrained HIV-1 envelope (Env)immunogens (i.e FLSC), therefore, made it feasible to induce antibodiessimilar to those used in the above cited infusion studies that affordprotection against HIV-1 [66–70]. Taken together, these observationsindicate that immunization with rdsRP-FLSC has the potential to induceneutralizing antibodies against primary isolates of HIV-1 and provideprotection against HIV-1 infection in humans.

EXAMPLE 4 Construction of an Anthrax rdsRP Vaccine

A functional eukaryotic translation expression cassette is obtained byincorporating an IRES that is functionally linked to the N-terminalregion (i.e. amino acids 10 to 254) of Bacillus anthrax lethal factor(herein designated “tLF”) by placing sequences encoding this immunogendownstream of the IRES in expression vector prφ8Seg-S (Example 2). Thesequence encoding tLF is obtained from pCLF4 ([73]; kindly provided byDr. Darrell Galloway, Department of Micribiology, Ohio State UniversityOhio) by PCR so that MscI and NotI sites are created at the 5-prime and3-prime ends, respectively (Example 1). The PCR-generated tLF fragmentis digested with MscI (New England Biolabs) and NotI (New EnglandBiolabs) and inserted, using T4 DNA ligase (New England Biolabs), intoMscI-, NotI-digested prφ8Seg-S, thereby functionally linking tLF to theIRES. The resultant plasmid is designated prdsRP-tLF and rdsRP thatincorporate the recombinant segment-S expressed by prdsRP-tLF (Example7) bear the capacity to express this non-toxic anthrax immunogen inmammalians cells. A second, functional eukaryotic translation expressioncassette is obtained by incorporating an IRES that is functionallylinked to the N-terminal region (i.e. amino acids 175 to 735) ofBacillus anthrax protective antigen (herein designated “tPA”) by placingsequences encoding this immunogen [73] downstream of the IRES inexpression vector prφ8Seg-S (Example 2). The sequence encoding tPA isobtained from pCPA ([73]; kindly provided by Dr. Darrell Galloway,Department of Micribiology, Ohio State University Ohio) by PCR so thatMscI and NotI sites are created at the 5-prime and 3-prime ends,respectively (Example 1). The PCR-generated tPA fragment is digestedwith MscI (New England Biolabs) and NotI (New England Biolabs) andinserted, using T4 DNA ligase (New England Biolabs), into MscI-,NotI-digested prφ8Seg-S, thereby functionally linking tPA to the IRES.The resultant plasmid is designated prdsRP-tPA and rdsRP thatincorporate the recombinant segment-S expressed by prdsRP-tPA (Example7) bear the capacity to express this anthrax immunogen in mammalianscells.

It is important to note that nucleic acid vaccines encoding tLF and tPAafforded protection in mice challenged intravenously with 5×50% lethaldoses of Bacillus anthrax lethal toxin (PA plus LF) [73]. In this study,100% of mice immunized with nucleic acid vaccine that expressed tLFalone, tPA alone, or the combination of both survived such a challenge,whereas all of the unvaccinated mice died [73]. Since neutralization ofBacillus anthrax toxin is a correlate of protection in humans, theseresults indicate that immunization with prdsRP-tLF and prdsRP-tPA aloneor in combination has the potential to induce Bacillus anthraxneutralizing antibodies and provide protection against a lethal Bacillusanthrax toxin infection in humans.

EXAMPLE 5 Construction of a rdsRP that Expresses an Immunogen and anAdjuvant

As an additional parallel track, the immunogenicity of rdsRP-1 (Example2) and rdsRP-2 (Example 6) can be enhanced significantly be includingsequences that encode the catalytic domain of cholera toxin (hereinreferred to as “ctxA1”), which are incorporated into a recombinantsegment-M in the rdsRP. To this end, a second PSA (i.e. thekanamycin-resistance gene herein designated “kan^(r)” from plasmidpUC18K1 [47] is inserted immediately downstream of the segment-M pacsequence, the latter being amplified from pLM2669, which encodes andexpresses a full-length cDNA copy of φ-8 segment-M (kindly provided byDr. Leonard Mindich). The CtxA1 gene functionally linked to the 53 bphepatitis C virus IRES (Genebank accession no. 1KH6_A; [45,46]) is theninserted downstream of kan^(R) by blunt-end ligation. The 53 bphepatitis C virus IRES is made synthetically (Example 1). Downstream ofthe ctxA1 gene, DNA sequences encoding a poly-adenylation site (frompcDNA3.1_(ZEO); See Example 1) and the 3-prime RNA-dependent RNApolymerase recognition sequence are included (the latter is amplifiedfrom pLM2669).

EXAMPLE 6 Introduction of an Alphavirus Amplicon into the rdsRP System

As noted above, rdsRP can harbor a mammalian translation expressioncassette comprised of Semliki Forest virus (herein referred to as “SFV”)self-amplifying replicon from plasmid pSFV1 (Invitrogen Inc., CarlsbadCalif.) functionally linked to syngp120 or to FLSC (See Examples 1 and2). Genes encoding SFV non-structural proteins (herein referred to as“NSPs” 1–4 and the replicase recognition site in pSFV1 are amplified byPCR and inserted by blunt-end ligation into the MscI site immediatelydownstream and functionally linked to the IRES in prφ8Seg-S (Example 2),resulting in prφ8Ampl-S (FIG. 6). Incidentally, the SmaI RE site inplasmid prφ8Ampl-S can serve as an insertion site for any desired gene,such as those outline above in the detailed description of theinvention. In this instance, however, PCR-generated DNA encoding thesyngp120 gene in pOGL1 (Example 1) is inserted into the SmaI site inprφ8Ampl-S, which places it immediately downstream of, and functionallylinked to, the SFV virus replicase recognition site (FIG. 6). RdsRP thatharbor this recombinant segment-S are designated herein as rdsRP-2.

EXAMPLE 7 Generation, Isolation and Purification of rdsRP-1 and rdsRP-2

Batches of rdsRP-1 and rdsRP-2 are generated by replicating a parentdsRP on the bacterial transformant the carries the expression prdsRP-1(i.e. expresses the5′-pacS-gene-8::PBAD-Ωasd::IRES::syngp120::poly-A::3′-Seg-S recombinantsegment-S; (wherein “::” indicates a novel nucleic acid junction;construction details are provided in Example 2) and prdsRP-2 (i.e.expresses the5′-pacS-gene-8::P_(BAD)-Ωasd::IRES::SFV_(nsp1-4)::syngp120::poly-A::3′-Seg-Srecombinant segment-S; Example 6), respectively (FIG. 4). Standardmethods for incorporation of recombinant segments into dsRP and thesubsequent replication, isolation and purification of the resultantrdsRP are used, as published in detail elsewhere [17,20,74,75] [14–16].Briefly, rdsRP are generated in Escherichia coli strain JM109;recombinant plasmids prdsRP-1 and prdsRP-2 are introduced into E. coliΔasd mutant strain χ6212 by transformation [76] and ampicillin-resistanttransformants are isolated on LBA containing 100 μg/ml ampicillin(Sigma).

The bacterial isolates are cultured at 37° C. for 24 hr; colonies thatgrow on the selective solid media are subsequently isolated and purifiedby standard methods [76]. To verify that the antibiotic-resistantisolates carrying the plasmid of interest, individual isolates arecultured in Luria-Bertani broth (LB; Difco, St Louis Mo.). Thetransformants are harvested after cultures reach an optical density at600 nm (OD₆₀₀) of 0.9, relative to the OD₆₀₀ a sterile LB control.Plasmid DNA is isolated from these cultures and analyzed by RE digestionusing those that generate a defined digestion pattern based on thepredicted sequence of the recombinant plasmid, including EcoRI, PstI,HindIII, HaeI, SmaI, NotI, and SalI. In addition, the plasmids arescreened by PCR using primers that amplify defined fragments within therecombinant segment-S including asd, IRES and syngp120. The PCR primersfor the amplifications are designed as outlined in Example 1. Theproducts of RE digestion and the PCR were analyzed by agarose gelelectrophoresis [76]. A positive clone is defined as one that displaysthe appropriate RE pattern and PCR pattern. Plasmids identified throughthis procedure can be further evaluated using standard DNA sequencingprocedures, as described (Example 1).

Finally, replication of parent dsRP φ-8 on χ6212 transformants thatharbor the recombinant plasmids prdsRP-1 or pdsRP-3 generates the rdsRPdesignated rdsRP-1 and rdsRP-3, respectively. χ6212 carriers of rdsRP-1and rdsRP-2 are isolated from within the resultant turbid plaques. Theselatter isolates are cultured on media lacking diaminopalmelic acid;under these circumstances only χ6212(rdsRP-1) and χ6212(rdsRP-2)carriers are capable of growth due to complementation of the lethal Δasdmutation by the expression of the recombinant segments in the rdsRPs.Methods for isolation and purification of rdsRP nucleocapsids, entailingliquid culture of carrier strain χ6212(rdsRP-1) and χ6212(rdsRP-2),osmotic lysis of the χ6212(rdsRP-1) and χ6212(rdsRP-2) bacilli andsucrose density gradient purification of the rdsRP-1 and rdsRP-2nucleocapsids, have been published extensively in detail by others[14–17,20,74,75]. Residual endotoxin is removed by adsorption to End-X®Endotoxin Affinity Resin (Cape Cod Associates Inc, Cape Cod Mass.). Thepurified rdsRP are placed into Spectrapore 50,000 Da cutoff dialysistubing and dialyzed in phosphate buffered saline (PBS) pH 7.3. Thenumber of plaque-forming units (pfu) in the nucleocapsid preparations ismeasured by infecting χ6212 protoplasts with 10-fold serial dilutions ofeach preparation and plating this suspension in soft-agar, as described[20]. The nucleocapsid concentration is adjusted to 5×10¹⁰ pfu/ml.

EXAMPLE 8 Infection of Human Dendritic Cells In Vitro with rdsRP

DsRP nucleocapsids have the unusual property of being able toauto-transform bacterial protoplasts, a process that requires gene-8[20,77]. Since the mechanism of protoplast transfection resembles thatof mammalian cells, rdsRP have the capacity to enter and expresspassenger immunogens in vitro following treatment of humanmonocyte-derived dendritic cells (MDDCs) with the purified rdsRP. Inshort, human PBMCs are separated from the blood of healthy donors bycentrifugation in Histopaque 1077 (Sigma, St. Louis, Mo.). The cells areenriched for monocytes (90–95% pure) using the StemSep® MonocyteEnrichment Cocktail and a magnetic negative-selection column (StemSep,Vancouver, British Columbia). Following enrichment, the monocytes areplated in RPMI 1640 (Gibco-BRL, Grand Island, N.Y.) and incubated for 2hours at 37° C. in a 5% CO₂ environment. Non-adherent cells and mediaare removed, and replaced with complete DC media, which comprises ofRPMI 1640 supplemented with 10% fetal bovine serum (Gibco-BRL), 1%sodium pyruvate (Sigma), 1% non-essential amino acids (Gibco-BRL),Gentamycin (Gibco-BRL), 50 μM β-mecaptoethanal (Sigma), 10 μM Hepes(Sigma), 35 ng/ml interleukin-4 (IL-4, R&D Systems, Minnesota, Minn.),and 50 ng/ml granulocyte/monocyte-colony stimulating factor (GM-CSF, R&DSystems). The cells in such cultures develop the appearance and cellsurface phenotype of immature MDDCs after 4 days in culture, asconfirmed by microscopy and flow cytometry.

To evaluate the delivery and expression of gp120 encoded in rdsRP-1,MDDCs are treated with a range of doses (from 10^(3–10) ⁷ pfu). Cellstreated with the rdsRP vectors and the control cells are harvested after24, 48 and 72 hr at 37° C. in 5% CO₂. The cells are washed twice withPBS and lysed in 1×SDS sample buffer and run on SDS-PAGE gels made with5% to 15% gradients of polyacrylamide. The samples are run undernon-reducing and reducing conditions to estimate the yields ofoligomeric forms of gp160. The samples are transferred to PVDFmembranes, which is probed with a mixture of monoclonal antibodiesspecific for defined epitopes of gp120 [66,78]. The extent ofglycosylation of Env proteins is estimated by treatment with Endo-Hprior to separation and evidence of glycosylation is taken as sine quanon that the gp120 RNA was expressed in the eukaryotic cell.

This experiment is designed to demonstrate that rdsRP-1 and rdsRP-2 bearan innate ability to enter mammalian cells and express gp120, whereinrdsRP-2 is capable of expressing significantly higher levels of gp120that rdsRP-1 due to the incorporation of the SFV amplicon in rdsRP-2(Example 6).

EXAMPLE 9 Immunogenicity of rdsRP Vaccine Vectors in Mice

Female BALB/c and C57B1/6 mice aged 6–8 weeks are obtained from JacksonLaboratories River (Bar Harbor, Me.). All mice are certifiedspecific-pathogen free and upon arrival at the University of MarylandBiotechnology Institute Animal Facility are maintained in amicroisolator environment and allowed to fee and drink ad lib.

The immunogenicity of rdsRP-1 (Example 2) and rdsRP-2 (Example 6) isassessed in groups of 10 mice, initially at dose of 10⁹ pfu. BothrdsRP-1 and rdsRP-2 are administered intragastrically three times spacedby 4-week intervals. In addition, a group of 10 mice is vaccinatedintranasally with three 10⁹-pfu doses of rdsRP-1 and a second similarsized group of mice is vaccinated with rdsRP-2; in both instances thedoses are spaced by 4-week intervals. In parallel, groups of 10 mice arevaccinated with a single 10⁹ pfu-dose of the rdsRP-1 or rdsRP-2,followed by two subcutaneous 50 μg-doses of soluble gp120 (or FLSC whenappropriate). This enables the assessment of rdsRP-1 as a primingvaccine.

Fully glycosylated gp120 used in such boosts is purified from serum-freeculture supernatants collected from 293 cells that are stablytransfected with pOGL1 (Expresses HIV-1_(MN) gp120) or pBaHu-120(Expresses HIV-1_(BaL1) gp120) and is supplied on a fee-for-servicebasis by the IHV μQuant core facility.

Additional groups of 10 mice are vaccinated intramuscularly with 10³ to10⁸ rdsRP-1 or rdsRP-1 pfu (in 10-fold serial dilutions) suspended inendotoxin-free saline (0.85% (w/v) NaCl), by direct injection using a30-gauge needle and a 1 ml tuberculin syringe. Booster vaccinations aregiven using the same formulation, route and dose as used to prime themice, spaced by 4-week intervals.

The immune-priming properties of each construct is determined bysacrificing groups of 5 mice 28 days after vaccination and the numbersof gp120-specific antibody secreting and CD4⁺ T cells are assessed asdescribed in Example 10. The remaining 5 mice in each group are boostedas delineated above.

When rdsRP-1 and rdsRP-2 prove adept at delivering inducing humoralresponses to the passenger immunogen, gp120, it will be possible toreduce the number of rdsRP-1 and rdsRP-2 dose. Thus, in the experimentalprotocol groups of 10 BALB/c mice that receive a single dose and twodoses of each test rdsRP are included. These groups assess theeffectiveness of both the prime and boosts in the extended three doseprotocols.

When the boosts prove unnecessary, the immunogenicity of 3-fold serialdilutions of each rdsRP, from 1×10⁴ to 1×10⁹ pfu, are evaluated todetermine whether the lower doses elicit immune responses to gp120 (SeeExample 10).

This series of vaccination experiments is designed to demonstrate thatrdsRP-1 and rdsRP-2 bear an innate ability to induce immune responses togp120 in mice vaccinated intragastrically, intranasally, andsubcutaneously. Since rdsRP-2 is capable of expressing significantlyhigher levels of gp120 that rdsRP-1 due to the incorporation of the SFVamplicon in rdsRP-2 (Example 6), the immune responses in mice vaccinatedwith rdsRP-2 are generally stronger in magnitude.

EXAMPLE 10 Measurement of Immune Responses

To measure serum IgG and IgA responses to gp120, sera are collectedbefore and at 10-day intervals after vaccination. About 400–500 μl ofblood is collected into individual tubes from the tail vein of eachmouse and allowed to clot by incubating for 4 hr on ice. Aftercentrifugation in a microfuge for 5 min, the sera are transferred tofresh tubes and stored at −80° C. Mucosal IgG and IgA responses to gp120are determined using fecal pellets and vaginal washes that are harvestedbefore and 10-day intervals after vaccination [79,80].

Standard ELISAs are used to quantitate the IgG and IgA responses togp120 in the sera and mucosal samples [78,81], and conducted as afee-for-service by the IHV's Viral Immunology Core facility. Fullyglycosylated gp120 for the ELISA assays is purified as described(Example 9). The purified gp120 is suspended in PBS at a concentrationof 3–10 μg/ml and will be used to coat 96-well ELISA plates. Ovalbuminis included in each ELISA as a negative control antigen and purifiedrdsRP nucleocapsids are included as a control antigen for vectorimmunogenicity. In addition, each ELISA also includes a positive controlserum, fecal pellet or vaginal wash sample, when appropriate. Thepositive control samples are harvested from mice vaccinated intranasallywith 10 μg gp120 mixed with 10 μg cholera toxin, as described [82]. Theend-point titers are calculated by taking the inverse of the last serumdilution that produced an increase in the absorbance at 490 nm that isgreater than the mean of the negative control row plus three standarderror values.

When a vaccine construct induces high-titer serum IgG and IgA responses,the gp120-specific IgG and IgA responses are also measured in themucosal compartment. Serum dimeric IgA is transported across mucosalsurfaces in mice and it is difficult to distinguish between locallyproduced IgA and systemically produced serum IgA in the mucosalsecretions. Therefore, a more direct measure of mucosal humoral immunityto gp120 is obtained by harvesting lamina propria lymphocytes from smallintestinal, colonic and vaginal tissue 40 and 80 days after vaccination,using procedures that preserve lymphocyte function [38,83]. IgA-specificELISPOT assays are conducted as described previously by our grouppreviously [38,83] and the results are expressed as the number ofgp120-specific IgA-producing cells per 10,000 IgA-producing cells [38].

When measuring the primary CD4⁺ T cell immune responses after the firstvaccination, groups of 5 mice are sacrificed 28 days after immunization,and Peyer's patch, lamina propria (mucosal sites) and spleen (systemicsite) cells are harvested using standard procedures [38,83]. Single cellsuspensions of enriched CD4⁺ T cells from these tissues are usedimmediately to measure the magnitude of the gp120-specific CD4⁺ T cellresponses by cytokine-specific ELISPOT assay [38]. Each sample isstimulated with three doses (0.1, 1.0 and 10) μg/ml of gp120 and thenumbers of gp120-specific CD4⁺ T cells are determined bycytokine-specific ELISPOT assays for IL-2, IL-4, IL-5, IL-6, IL-10 andIFN-γ production. All ELISPOT assays are conducted usingcommercially-available capture and detection mAbs (R&D Systems andPharmingen), as described [84,85]. Each assay includes mitogen (Con A)and ovalbumin controls.

EXAMPLE 11 Vaccination Protocol Discrimination Criteria

As indicated in example 10, the magnitude of humoral and CD4⁺ T cellresponses to the selected HIV-1 immunogens in mice vaccinatedintragastrically and intranasally with the experimental rdsRP constructsare measured by conventional ELISA and ELISPOT assays. Individual immuneresponse parameters are evaluated quantitatively with the idea ofcharacterizing the magnitude and duration of the host responses that aregenerated by each construct. In addition, all experimental values aremeasured in triplicate and standard statistical analyses are used whenmeasuring and comparing the individual responses (including ANOVA andStudent T tests). To ensure reproducibility, each experiment isperformed a minimum of two times. When appropriate, the number of micecan be increase in individual groups if trends are observed but there isinsufficient statistical power to resolve the differences. The followingset of criteria was formulated to enable one to discriminate between therdsRP vaccination protocols in example 9:

“Go” Criteria:

-   -   1. The location of the response: Preference is assigned to        vaccination protocols that elicit gp120-specific humoral        responses in both mucosal and systemic sites.    -   2. The magnitude of the responses: Preference is assigned to        vaccination protocol that elicits the strongest gp120-specific        antibody and/or antibody secreting cell responses.    -   3. The duration of the response: Preference is assigned to        vaccines that elicit responses that remain significantly        elevated for the longest period after vaccination.    -   4. The minimum effective dose: Preference is assigned to        vaccination protocols that achieve the immune responses above        with the minimum dose of rdsRP and the fewest doses.        “No-Go” Criteria:    -   1. Vaccination protocols that fail to immune responses to the        passenger immunogen.    -   2. When pertinent (i.e. when FLSC immunogens are inserted into        the rdsRP instead of gp120), vaccination protocols that fail to        induce broadly neutralizing antibodies to primary HIV-1        isolates.

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1. A double stranded RNA (dsRNA) phage that expresses at least onegenetic sequence in eukaryote cells, comprising: a cap independenttranslation enhancer (CITE); and at least one genetic sequence that isexpressed in a eukaryote cell, wherein said CITE and said at least onegenetic sequence are functionally linked and are incorporated into oneor more dsRNA segments in the dsRNA.
 2. The dsRNA phage of claim 1wherein said at least one genetic sequence encodes a vaccine antigen. 3.The dsRNA phage of claim 1 wherein said at least one genetic sequenceencodes a bioactive protein.
 4. The dsRNA phage of claim 1 wherein saidat least one genetic sequence encodes an immunoregulatory protein. 5.The dsRNA phage of claim 1 wherein said at least one genetic sequenceencodes an antisense RNA.
 6. The dsRNA phage of claim 1 wherein said atleast one genetic sequence encodes a catalytic RNA.
 7. The dsRNA phageof claim 1 wherein said at least one genetic sequence encodes animmunogen.
 8. The dsRNA phage of claim 1 wherein said CITE and said atleast one genetic sequence are incorporated into an L segment.
 9. ThedsRNA phage of claim 1 wherein said CITE and said at least one geneticsequence are incorporated into an M segment.
 10. The dsRNA phage ofclaim 1 wherein said CITE and said at least one genetic sequence areincorporated into an S segment.
 11. The dsRNA phage of claim 1 whereinat said at least one genetic sequence includes a sequence encoding forgreen fluorescent protein.
 12. The dsRNA phage of claim 1 wherein saidat least one genetic sequence encodes an immunogen and a cytokine. 13.The dsRNA phage of claim 1 further comprising an alpha-virusself-amplifying expression system.
 14. The dsRNA phage of claim 7wherein said immunogen is viral.
 15. The dsRNA phage of claim 7 whereinsaid immunogen is bacterial.
 16. The dsRNA phage of claim 7 wherein saidimmunogen is from a parasite.
 17. The dsRNA phage of claim 7 whereinsaid immunogen is a therapeutic agent.
 18. The dsRNA phage of claim 7wherein said immunogen is an autoimmune antigen.
 19. The dsRNA phage ofclaim 7 wherein said immunogen is a tumor antigen or tumor specificantigen.
 20. The dsRNA phage of claim 13 wherein said alpha-virusself-amplifying expression system is based on semliki forest virus.